MeCP2 (methyl CpG Binding Protein 2) is a 53 kDa nuclear protein that can repress transcription, regulate RNA splicing, and modulate chromatin architecture. Mutations in MeCP2 have been causally linked to the debilitating neurological disorder, Rett Syndrome (RTT). The current view is that MeCP2 is a multifunctional nuclear regulatory protein with important roles in health and disease. My laboratory studies how MeCP2 affects chromatin structure and function. During the last grant period we demonstrated that MeCP2 functions to direct both local fiber condensation and global fiber-fiber interactions when bound to both methylated and unmethylated chromatin fibers. In addition, comprehensive biochemical characterization of the purified recombinant protein was completed. Surprisingly, MeCP2 is a monomer in solution, is 60% intrinsically disordered and ~35% 2-sheet/turn, and has a random coil-like tertiary structure. Further, MeCP2 is organized into six trypsin resistant domains, each of which appears to contain one or more long segments of intrinsic disorder. These advances raise many new questions related to my long-term objective of understanding MeCP2 structure and how it is linked to MeCP2 multifunctionality and dysfunction in RTT. To address this objective, I propose to the following Specific Aims: (1) to dissect the tertiary structure of MeCP2 in solution by studying the structure of the individual domains, the role of intrinsic disorder, and specific MeCP2 RTT mutants, (2) to characterize the mechanism of MeCP2 interaction with dsDNA and mononucleosomes by identifying and analyzing the multiple dsDNA and chromatin binding sites in MeCP2 and determining the structural changes that occur in MeCP2 upon DNA binding, and (3) to establish the pathway through which MeCP2 assembles condensed supramolecular nucleoprotein complexes in vitro by determining the sequence of molecular events involved in MeCP2-dependent chromatin fiber condensation. The proposed studies will yield important advances at both the protein, nucleosome, and chromatin fiber levels. The proposed research is innovative in the magnitude and scale of the structures being studied;the MeCP2 monomer is 53 kDa, the MeCP2-nucleosome complex exceeds 300 kDa, and the MeCP2-chromatin fiber suprastructures can be several hundred megadaltons. Nevertheless, all of these structures will be studied in vitro as pure recombinant components. The proposed experiments are driven by numerous specific hypotheses, and will be among the first to dissect a structurally perplexing, disease-related, intrinsically disordered protein. Collectively, these studies will provide an unprecedented body of information that is directly relevant to MeCP2 structure, will yield the first rigorous understanding of how MeCP2 influences genome structure and function, and will begin to lay a structural foundation for understanding the role of MeCP2 in the molecular pathogenesis of RTT. PUBLIC HEALTH RELEVANCE: Mutations in the MeCP2 protein are causative of a debilitating neurological disorder, Rett Syndrome, which is the most common inherited mental retardation syndrome. The proposed experiments will greatly increase our understanding of the structure of MeCP2, and how it functions as a chromatin architectural protein. In doing so, these studies will lay a biochemical foundation for understanding the role of MeCP2 in the molecular pathogenesis of RTT.